Anemia, a common condition in Veteran patients, is a potent risk factor for increased morbidity and mortality when accompanying other disease states. Although therapies for anemia have improved over the past several decades, almost all current therapies involve bolus administration of erythropoietin (epo), an endocrine factor produced in the adult kidney and also in the liver with severe anemia. Non-physiological, bolus epo administration has untoward effects, including a possible increased thrombotic risk as well as an undesirable stimulatory effect on cancer. Understanding how epo is regulated will allow investigators to develop rational therapies besides epo replacement. Our current focus is defining molecular mechanisms regulating epo expression in mammals. Hypoxia Inducible Factor (HIF) transcription factors are a family of molecular mediators that induce the protective cellular response to hypoxia. We were the first to establish that the second HIF member, HIF-2, is critical for in vivo epo gene expression. While an essential role of HIF-2 in epo regulation is now recognized, the factors responsible for temporal epo gene expression in vivo, or for abnormal repression of epo gene expression in anemia patients, remain poorly understood. We recently identified a novel mechanism of HIF-2 signaling that affects epo regulation. Activity of HIF-1, the founding HIF member, is controlled predominantly by changes in protein levels, which is mediated by oxygen-dependent post-translational modifications of the HIF-1 protein. However, although the HIF-2 protein undergoes the same oxygen-dependent post-translational modifications, this mechanism is not the sole or even major mechanism for controlling HIF-2 signaling. If not the lack of oxygen, what then is the molecular trigger for activating HIF-2? Our central hypothesis is that hypoxia triggers changes in intermediary metabolism to effect acetylation of HIF-2. In Preliminary Data, we show that HIF-2 activity is controlled by two post-translational modifications, acetylation and deacetylation. In Pilot Studies, we identify the molecular and biochemical basis for HIF-2 acetylation, termed the acetate switch, which involves changes in intermediary metabolism to induce HIF-2 acetylation and coactivator recruitment. Deciphering how the acetate switch regulates HIF-2 signaling will provide novel insights into normal epo regulation and will identify selective molecular targets for modulation of endogenous epo gene expression in patients with anemia.